Characterization of the metal ion binding site in the anti-terminator protein, HutP, of Bacillus subtilis

HutP is an RNA-binding protein that regulates the expression of the histidine utilization (hut) operon in Bacillus subtilis, by binding to cis-acting regulatory sequences on hut mRNA. It requires L-histidine and an Mg(2+) ion for binding to the specific sequence within the hut mRNA. In the present study, we show that several divalent cations can mediate the HutP–RNA interactions. The best divalent cations were Mn(2+), Zn(2+) and Cd(2+), followed by Mg(2+), Co(2+) and Ni(2+), while Cu(2+), Yb(2+) and Hg(2+) were ineffective. In the HutP–RNA interactions, divalent cations cannot be replaced by monovalent cations, suggesting that a divalent metal ion is required for mediating the protein–RNA interactions. To clarify their importance, we have crystallized HutP in the presence of three different metal ions (Mg(2+), Mn(2+) and Ba(2+)), which revealed the importance of the metal ion binding site. Furthermore, these analyses clearly demonstrated how the metal ions cause the structural rearrangements that are required for the hut mRNA recognition

Insights into anti-termination regulation of the hut operon in Bacillus subtilis: importance of the dual RNA-binding surfaces of HutP

The anti-termination protein, HutP, regulates the gene expression of the hut (histidine utilization) operon of Bacillus subtilis, by destabilizing the hut terminator RNA located upstream of the coding region encoding l-histidine degradation enzymes. On the basis of biochemical, in vivo and X-ray structural analyses, we now report that HutP uses its dual RNA-binding surfaces to access two XAG-rich regions (sites I and II) within the terminator RNA to mediate the destabilization process. In this process, HutP initiates destabilization at the 5′-end of its mRNA by binding to the first XAG-rich region (site I) and then accesses the second XAG-rich region (site II), located downstream of the stable G-C-rich segment of the terminator stem. By this action, HutP appears to disrupt the G-C-rich terminator stem, and thus prevents premature termination of transcription in the RNA segment preceding the regions encoding for the histidine degradation enzymes

Crystal structure of glutamine receptor protein from Sulfolobus tokodaii strain 7 in complex with its effector l-glutamine: implications of effector binding in molecular association and DNA binding

Genome analyses have revealed that members of the Lrp/AsnC family of transcriptional regulators are widely distributed among prokaryotes, including both bacteria and archaea. These regulatory proteins are involved in cellular metabolism in both global and specific manners, depending on the availability of the exogenous amino acid effectors. Here we report the first crystal structure of glutamine receptor protein (Grp) from Sulfolobus tokodaii strain 7, in the ligand-free and glutamine-bound (Grp-Gln) forms. Although the overall structures of both molecules are similar, a significant conformational change was observed at the ligand [l-glutamine (Gln)] binding site in the effector domain, which may be essential for further stabilization of the octameric structure, and in turn for facilitating DNA binding. In addition, we predicted promoter for the grp gene, and these analyses suggested the importance of cooperative binding to the protein. To gain insights into the ligand-induced conformational changes, we mutated all of the ligand-binding residues in Grp, and revealed the importance of Gln binding by biochemical and structural analyses. Further structural analyses showed that Y77 is crucial for ligand binding, and that the residues T132 and T134, which are highly conserved among the Lrp family of proteins, fluctuates between the active and inactive conformations, thus affecting protein oligomerization for DNA binding

ST1710–DNA complex crystal structure reveals the DNA binding mechanism of the MarR family of regulators

ST1710, a member of the multiple antibiotic resistance regulator (MarR) family of regulatory proteins in bacteria and archaea, plays important roles in development of antibiotic resistance, a global health problem. Here, we present the crystal structure of ST1710 from Sulfolobus tokodaii strain 7 complexed with salicylate, a well-known inhibitor of MarR proteins and the ST1710 complex with its promoter DNA, refined to 1.8 and 2.10 Å resolutions, respectively. The ST1710–DNA complex shares the topology of apo-ST1710 and MarR proteins, with each subunit containing a winged helix-turn-helix (wHtH) DNA binding motif. Significantly large conformational changes occurred upon DNA binding and in each of the dimeric monomers in the asymmetric unit of the ST1710–DNA complex. Conserved wHtH loop residues interacting with the bound DNA and mutagenic analysis indicated that R89, R90 and K91 were important for DNA recognition. Significantly, the bound DNA exhibited a new binding mechanism

Purification, crystallization and preliminary X-ray crystallographic analysis of ST1022, a putative member of the Lrp/AsnC family of transcriptional regulators isolated from Sulfolobus tokodaii strain 7

A putative member of the Lrp/AsnC family of transcriptional regulators, ST1022 from S. tokodaii strain 7, has been purified and crystallized in the absence and presence of the effector l-glutamine. A molecular-replacement solution was found using the FL11 transcriptional regulator from Pyrococcus sp. OT3 as a model and structural refinement is under way